This invention pertains to a method and a device to assay chromatographic eluate for antigens of interest where affinity chromatography is used in parallel with a control.
An antigen is a chemical or biological substance capable of eliciting production of antibodies when it is introduced into a responding organism. The organism, once challenged by the antigen, responds with the immunological production of structurally complementary antibodies. These antibodies then attack the specific antigen because a particular antibody binds only to those antigens complementary to its structure. Therefore, a particular antibody neglects the other, noncomplementary antigens.
Specifically binding pairs of antigens and antibodies are very useful laboratory reagents. Once isolated and purified, particular antibodies may be used to selectively bind to their complementary antigens due to the binding characteristic existing between them. The antibodies are usually tagged with radioactive or fluorescent labels and placed within a reaction vessel. Then, when exposed to a complementary antigen, the antigen-antibody complex forms and tags the presence of the antigen. These complexes may be separated from the reaction vessel and any antigen present is indirectly quantified on the basis of the label present in the complex.
Other uses of specifically binding pairs include competitive binding processes. Similar labels as those mentioned previously are first coupled to antigen molecules. The labelled antigens are then introduced to antibodies in a known amount. Afterward, a chemical or biological sample containing an unlabeled antigen is introduced to the system. The presence and quantity of the unlabeled antigen is then calculated based upon the competition for antibody binding sites using a known amount of labeled antigen to calculate an unknown amount of unlabeled antigen. Such techniques are well known in the art.
Furthermore, it is also known in the art to attach antibodies to a solid support. With attached antibodies, the support becomes a testing material capable of specifically binding labeled or unlabeled antigens. The presence of antigens may then be subsequently detected by monitoring labels as previously mentioned. The attachment of antibodies to a support allows the analytical use of antibodies to be moved from the confines of a reaction vessel to an open system. Particularly, supported antibodies may reside within a continuous flow of effluent, such as, that found in liquid chromatography.
Liquid chromatographic techniques are well known for separating chemical or biological compounds into distinct zones within a suitable eluant. Once separated, analytes may be isolated and further steps may be taken to identify a particular species. However, to avoid the isolation step and to obtain an in-process identification, often an analytical technique such as measuring the eluate's ultraviolet light absorption is used.
In a typical ultraviolet light absorption scheme, a chart recorder or other device continuously records the transmission of ultraviolet light through the flow path of the eluate. When a detectable species enters the detector, the absorption changes from a baseline, the baseline being the absorption seen when only the eluant is present. This change, termed a "peak", marks the passage of a chemical or biological species through the detector. Given that retention time within a chromatographic column is somewhat species specific, the time required for a particular species to flow through the chromatograph should be constant for a particular flow rate of eluant. Therefore, an unknown species may be identified by comparing the retention time of a sample with that of a known and suspected species. However, this method has limitations.
The ultraviolet light absorption scheme directly described above is not applicable in all situations. First, in order to calculate a retention time, an injection or start time must be known. If it is desired to simply monitor a continuous effluent flow; the above scheme would not work because it is not known when a suspected analyte enters the system. A second problem occurs where multiple species have overlapping retention times. It may be particularly difficult to tie a particular species with a particular peak when it may be masked by other chemical species. A third situation occurs when a retention time may slightly change for various reasons. This may result in a false positive or negative when the experimental data is compared to the prior standard.
What is needed is a method and a device that detects "on-line" the presence of a specific antigen without requiring the calculation of retention times, and that is usable in the presence of other chemical or biological species.